Mixing of fluids in fluidic systems

ABSTRACT

Fluidic devices and methods associated with mixing of fluids in fluidic devices are provided. In some embodiments, a method may involve the mixing of two or more fluids in a channel segment of a fluidic device. The fluids may be in the form of, for example, at least first, second and third fluid plugs, composed of first, second, and third fluids, respectively. The second fluid may be immiscible with the first and third fluids. In certain embodiments, the fluid plugs may be flowed in series in the channel segment, e.g., in linear order, causing the first and third fluids to mix without the use of active to components such as mixers. The mixing of fluids in a channel segment as described herein may allow for improved performance and simplification in the design and operations of fluidic devices that rely on mixing of fluids.

RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 61/778,905, filed Mar. 13, 2013, andentitled “Mixing of Fluids in Fluidic Systems,” which is incorporatedherein by reference.

FIELD OF INVENTION

The present embodiments relate generally to methods for flowing fluidsin fluidic devices, and more specifically, to methods that involve themixing of fluids.

BACKGROUND

The manipulation of fluids plays an important role in fields such aschemistry, microbiology and biochemistry. These fluids may includeliquids or gases and may provide reagents, solvents, reactants, orrinses to chemical or biological processes. While various fluidic (e.g.,microfluidic) methods and devices, such as microfluidic assays, canprovide inexpensive, sensitive and accurate analytical platforms, fluidmanipulations—such as the mixture of multiple fluids, sampleintroduction, introduction of reagents, storage of reagents, separationof fluids, collection of waste, extraction of fluids for off-chipanalysis, and transfer of fluids from one chip to the next—can add alevel of cost and sophistication. Accordingly, advances in the fieldthat could reduce costs, simplify use, and/or improve fluidmanipulations in microfluidic systems would be beneficial.

SUMMARY OF THE INVENTION

Methods for flowing fluids in fluidic devices, and related components,devices and systems associated therewith are provided. The subjectmatter of this application involves, in some cases, interrelatedmethods, alternative solutions to a particular problem, and/or aplurality of different uses of fluids and devices.

In one set of embodiment, a series of methods are provided. In oneembodiment, a method comprises flowing in series in a channel a firstfluid plug comprising a first fluid, a second fluid plug comprising asecond fluid, and a third fluid plug comprising a third fluid. The firstfluid plug has a first volume. The second fluid plug is positionedbetween the first and third fluid plugs and the second fluid isimmiscible with each of the first and third fluids. The method furthercomprises reducing the first volume of the first fluid plug by at least50% and combining at least a portion of the first fluid into the thirdfluid plug so as to mix at least portions of the first and third fluids.

In another embodiment, a method comprises flowing in series in a channela first fluid plug comprising a first fluid, a second fluid plugcomprising a second fluid, and a third fluid plug comprising a thirdfluid. The second fluid is immiscible with each of the first and thirdfluids and the second fluid plug is positioned between the first andthird fluid plugs. The first fluid comprises a first component for achemical and/or biological reaction and the third fluid comprises asecond component for a chemical and/or biological reaction. The firstcomponent is different from the second component. The method furthercomprises depositing at least a portion of the first fluid on a wall ofthe channel during the flowing step and combining at least a portion ofthe first fluid deposited on the wall of the channel into the thirdfluid plug so as to mix at least portions of the first and third fluids.

In one embodiment, a method comprises flowing in series in a channel afirst fluid plug comprising a first fluid, a second fluid plugcomprising a second fluid, and a third fluid plug comprising a thirdfluid. The first fluid comprises a first component for a chemical and/orbiological reaction and the third fluid comprises a second component fora chemical and/or biological reaction. The second fluid is immisciblewith the first and third fluids, and the second fluid plug is positionedbetween the first and third fluid plugs. The method further comprisescombining at least a portion of the first fluid into the third fluidplug so as to mix at least portions of the first and third fluids andperforming one or more chemical and/or biological reactions involvingeach of the first and second components.

In another embodiment, a method comprises providing a fluidic devicecontaining a first fluid and a second fluid. The first and second fluidsare stored and sealed in the fluidic device and kept separate from oneanother during storage. The method further comprises unsealing thefluidic device and flowing in series in a channel a series of fluidplugs comprising a first fluid plug comprising the first fluid, a secondfluid plug comprising the second fluid, and a third fluid plugcomprising a third fluid. The third fluid plug is positioned between thefirst and second fluid plugs and the third fluid is immiscible with thefirst and second fluids. The method further comprises combining at leasta portion of the first fluid into the second fluid plug so as to mix atleast portions of the first and second fluids.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIGS. 1A-1E show methods of mixing fluid plugs in a channel segmentaccording to one set of embodiments;

FIGS. 2A-2D show methods of mixing multiple fluid plugs simultaneouslyin a channel segment according to one set of embodiments;

FIGS. 3A-3D show methods of mixing of at least three different fluidplugs in a channel segment according to one set of embodiments;

FIGS. 4A-4E show methods of mixing fluid plugs with a substantially dryreagent in a channel segment according to one set of embodiments;

FIGS. 5A-5B show cross-sectional dimensions of a channel segmentaccording to one set of embodiments;

FIGS. 6A-6C show methods of mixing involving the use of vent valvesaccording to one set of embodiments;

FIG. 7 shows a plot demonstrating the influence of hydraulic channeldiameter on mixing according to one set of embodiments;

FIG. 8 shows a plot demonstrating the influence of treated and untreatedchannels on mixing according to one set of embodiments;

FIGS. 9-11 show plots of proportions of solutions after serial mixingbetween multiple fluids according to one set of embodiments; and

FIGS. 12A-12C show channels including different draft angles accordingto one set of embodiments.

DETAILED DESCRIPTION

Fluidic devices and methods associated with mixing of fluids in fluidicdevices are provided. In some embodiments, a method may involve themixing of two or more fluids in a channel segment of a fluidic device.Mixing may take place when at least some of the fluids are positionedseries in the channel segment. The fluids may be in the form of, forexample, at least first, second and third fluid plugs, composed offirst, second, and third fluids, respectively. The second fluid may beimmiscible with the first and third fluids. In certain embodiments, thefluid plugs may be flowed in series in the channel segment, e.g., inlinear order. As the first fluid plug flows in the channel segment, atleast a portion of the first fluid may be removed from the first plug,thereby reducing the volume of the first fluid plug. For instance,portions of the first fluid may be deposited on the wall of the channelduring this flowing step. As the third fluid plug flows in the channel,the third fluid may mix with portions of the deposited fluid to form amixture of the first and third fluids in the third fluid plug. Themixing of fluids in a channel segment as described herein may allow forimproved performance and simplification in the design and operations offluidic devices that rely on mixing of fluids. For example, in someembodiments active components such as mixers are not needed in thefluidic device.

An example of a method of mixing in a channel segment is shown in FIGS.1A-E. As shown illustratively in FIG. 1A, a channel segment 5, includingan upstream portion 7 and a downstream portion 8, may contain a firstfluid plug 10-1 containing a first fluid 10-1A, a second fluid plug 20-2containing a second fluid 20-2A, and a third fluid plug 10-3, containinga third fluid 10-3A. As shown illustratively in this figure, the secondfluid plug may be positioned between and directly adjacent to the firstand third fluid plugs. In some embodiments, the second fluid may beimmiscible with the first and third fluids, while the first and thirdfluids may optionally be miscible with one another. For example, thesecond fluid may be a gas (e.g., air) and the first and third fluids maybe liquids. Other fluid plugs may also be present in the channel segmentas described in more detail below.

As used herein, when a fluid or fluid plug is referred to as being“adjacent” another fluid or fluid plug, it can be directly adjacent thefluid or fluid plug, or an intervening fluid or fluid plug also may bepresent. A fluid or fluid plug that is “directly adjacent” or “incontact with” another fluid or fluid plug means that no interveningfluid or fluid plug is present.

As shown in FIG. 1B, the fluids may be flowed in series, e.g., fromupstream to downstream in the direction of arrow 9. The channel segmentmay be configured such that the flowing of the fluid plugs leads to thereduction of volume of the first fluid plug. For example, at least aportion of the first fluid (e.g., fluid portion 10-1B) may deposit ontoa wall of the channel segment during fluid flow. Various channelconfigurations and methods for reducing the volume of the first fluidplug are described in more detail herein. In certain embodiments, inwhich the second fluid is immiscible with the first fluid, fluid portion10-1B does not combine with the second fluid plug and as the secondfluid plug flows in the channel segment. In embodiments in which thethird fluid is miscible with the first fluid, the first and third fluidsmay combine to form a mixture 10-3C of at least portions of the twofluids, as shown illustratively in FIG. 1C.

In some cases, as the first fluid plug flows, its volume may continue toreduce to a desired extent, for example, until mixture 10-3C includes acertain ratio of the first and third fluids, until a particular reducedvolume of the first fluid plug has been reached, until a particularconcentration of a component is present, or until a particular physicalor chemical property is achieved. In some cases, the volume of the firstfluid may be reduced by, for example, at least 50% as shown in FIG. 1C.In other cases, as shown illustratively in FIG. 1D, the entire volume ofthe first fluid plug may be reduced, such that only the second and thirdfluid plugs remain. The third fluid plug may then mix with the entirevolume of the first fluid, as shown in FIG. 1E.

In some embodiments, the first and third fluids may contain a first andsecond component, respectively, for a chemical and/or biologicalreaction. In some cases, the first and second components are the same.In other embodiments, the first and second components are different. Insome instances, a chemical and/or biological reaction involving thefirst and second components may be performed within the third fluid plugcontaining the mixture of the first and third fluids. For example, thefirst fluid may contain a silver salt and the third fluid may contain areducing agent. The mixture of the first and third fluid may react witha reagent (e.g., gold colloids) to form detectable species (e.g., asilver film or particles that may be detected, for example, optically),as described in more detail below. Additional examples of chemicaland/or biological reactions are described in more detail below. Incertain embodiments, one or more fluid plugs contains a rinse solution.Other types of fluids are also possible.

As described herein, in some embodiments a fluid from a fluid plug maybe deposited onto a wall of a channel (e.g., in the form a fluid portionwhich may be available for mixing with a fluid from another fluid plug).The fluid portion may be deposited as a film (e.g., a continuous ordiscontinuous film) of liquid on the wall of a channel, as fluiddroplets, or in any other suitable form. The form in which depositionoccurs may depend on factors such as the type of fluid being deposited,surface tension, surface energy of the channel wall, surface roughnessof the channel wall, channel geometry and/or other factors. In somecases, at least a portion of the fluid deposited on the wall remains onthe wall of the channel for the remainder of fluid flow. In other cases,however, substantially all of the fluid portion is combined with anotherfluid during subsequent fluid flow.

An example of a method of mixing several fluids in a channel segment isshown in FIGS. 2A-E. As shown in FIG. 2A, channel segment 5, includingupstream portion 7 and downstream portion 8, may contain multiple fluidplugs. In some embodiments, as illustrated in FIG. 2A, the channelsegment may include a first 10-1, a second 20-2, a third 10-3, a fourth20-4, a fifth 10-5, a sixth 20-6, a seventh 10-7, an eighth 20-8, aninth 10-9, a tenth 20-10, and an eleventh 10-11 fluid plug, whichcontain a first, a second, a third, a fourth, a fifth, a sixth, aseventh, an eighth, a ninth, a tenth, and an eleventh fluid,respectively. In some cases, the fluid plugs may alternate in respect toa particular property (e.g., phase, composition, viscosity, pH, volume,etc.). For example, in one set of embodiments, the odd numbered fluidsshown in FIG. 2 (i.e., first, third, fifth, seventh, ninth, andeleventh) may be liquids and the even numbered fluids (i.e., second,fourth, sixth, eighth, and tenth) may be immiscible with those liquids(e.g., they may be gases). It should be understood that the labeling of“odd” or “even” fluids is for descriptive purposes only and is notintended to limit the fluids to a particular property or configuration.For instance, in other embodiments, one or more odd numbered fluidsdescribed herein may be immiscible fluids (e.g., gases) and one or moreeven numbered fluids may be liquids. Other configurations are alsopossible.

In some embodiments, the channel segment may be configured such thatflowing the fluids through the channel segment results in the depositionof fluids from more than one fluid plug (e.g., odd numbered fluids) on awall of the channel segment, as shown illustratively in FIG. 2B. Thisdeposition may occur simultaneously or subsequently. As shown in FIG.2B, fluid portion 10-1B may be removed from fluid 10-1A and fluidportion 10-3B may be removed from fluid 10-3A, e.g., by the fluidportions being deposited on a wall of the channel segment (e.g.,dispersed along or within the channel). During flow, the fluid portionsmay mix with the next “like”-fluid upstream in the sequence. Forinstance, in embodiments in which the odd numbered fluids are misciblewith each other but immiscible with the even numbered fluids, the fluidportions (formed from an odd numbered fluid) may mix with other oddnumbered fluids and do not mix with the even numbered fluids. Forexample, fluid portion 10-5B from the fifth fluid plug may mix with thefluid in the seventh, but not the sixth, fluid plug. Simultaneously orsequentially, fluid portion 10-7B from the seventh fluid plug may mixwith the fluid in the ninth fluid plug, but not the eight fluid plug.

In some embodiments, as the fluids flow in series, the composition (orother property such as viscosity, pH, and/or volume) of the fluidportions and each fluid in its respective fluid plug may change, asillustrated in FIG. 2C. For instance, the third fluid plug may containfluid 10-3A at the start of the process, as shown in FIG. 2A. As thethird fluid plug flows, the third fluid may mix with (and optionallyreact with) fluid portion 10-1B from the first fluid to form a mixture10-3C of the first and third fluid in the third fluid plug. Subsequentfluid portions 10-3D removed from the third fluid plug may be a mixtureof the first and third fluid as shown in FIG. 2C-2D. In some cases, asthe fluids flow in series, the volume of the fluid in the first fluidplug may be reduced by various amounts. In certain cases, the entirevolume of a fluid (e.g., the first fluid as shown illustratively in FIG.2D) may be incorporated into one or more subsequent fluid plugs thatcontain fluids miscible with the fluid, such that the fluid plug is nolonger present in the channel segment.

Another example of a method of mixing several fluids in a channelsegment is shown in FIGS. 3A-D. As shown illustratively in FIGS. 3A-D,channel segment 5, including upstream portion 7 and downstream portion8, may contain multiple fluid plugs that alternate in respect toparticular property (e.g., phase), such that the fluid in each fluidplug is immiscible with the fluids in adjacent fluid plugs. Forinstance, as shown in FIG. 3A, first fluid 10-1A, third fluid 10-3Afifth fluid 10-5A, seventh fluid 10-7A, ninth fluid 10-9A, and eleventhfluid 10-11A are separated from each other by intervening fluid plugs20-2, 20-4, 20-6, 20-8, and 20-10. The first, third, and fifth fluidsmay differ in a particular property (e.g., composition, viscosity, pH,volume, etc.) and the seventh, ninth, and eleventh fluids also maydiffer in a particular property (e.g., composition, viscosity, pH,volume, etc.). In some embodiments, the first, third, and fifth fluidsmay have a particular property that is substantially similar to theseventh, ninth, and eleventh fluids, respectively, although in otherembodiments the particular property may differ. When flowed in thechannel segment, at least one of the first, third, fifth, seventh,ninth, and eleventh fluid plugs may deposit a fluid portion (e.g.,10-1B, 10-3B, 10-5B, 10-7B, 10-9B, 10-11B, respectively) on a wall ofthe channel, as illustratively shown in FIG. 3B. During flow, the fluidportions may mix with the next miscible fluid upstream in the sequence,as indicated by arrows 40 shown in FIG. 3B-C.

In some embodiments, a fluid, after mixing with a fluid portion, maybecome substantially different from a fluid in another fluid plug withrespect to at least one property (e.g., composition, viscosity, pH,volume, etc.). For instance, as shown in FIG. 3C, seventh fluid 10-7A,which may initially be substantially similar in composition to firstfluid 10-1A (e.g., prior to mixing), may differ from the first fluidafter mixing with a fluid portion (e.g., fluid portion 10-5B from fifthfluid 10-5A). In other embodiments, a fluid, after mixing with a fluidportion, may become substantially similar to a fluid in another fluidplug with respect to at least one property (e.g., composition,viscosity, pH, volume, etc.). For example, eleventh fluid 10-11A maybecome substantially similar to third fluid 10-3A after the eleventhfluid mixes with fluid portion 10-9B, which has the same composition asthe third fluid.

It should be appreciated that while FIGS. 3A-3D show that mixing canoccur between each “like” fluid of fluid plug (e.g., first, third,fifth, seventh, ninth, and eleventh fluids), in other embodiments, oneor more such fluids/fluid plugs may be designed to not mix with anotherfluid, e.g., by controlling surface tension, polarity, interfacialtension, and/or other factors as described in more detail herein. Forexample, in one embodiment, fifth fluid plug 10-5 may be designed suchthat fluid 10-5A within the fluid plug is not substantially removed fromthe fluid plug during fluid flow. In such an embodiment, fluid portion10-3B from the third fluid plug may flow past fluid plug 10-5 and maymix directly with fluid from seventh fluid plug 10-7. Otherconfigurations of mixing are also possible.

In certain embodiments, a fluid plug may contain fluids from more thanone fluid plug, e.g., after a mixing process described herein. Duringfluid flow, the fluid plug containing the multiple fluids may itselfhave fluid removed from it (e.g., by depositing fluid on a wall of achannel segment and/or dispersed along or within the channel) tofacilitate further mixing of fluids. For example, as illustrated in FIG.3C, the first, third, fifth, seventh, ninth, and eleventh fluid plugsmay contain miscible fluids. During flow, the first fluid plug 10-1 mayhave a fluid portion 10-1B removed from it, which mixes with the thirdfluid 10-3A in the third fluid plug 10-3. As the fluids continue to flowin the channel segment, the third fluid plug 10-3 may have a fluidportion 10-3D (i.e., a mixture of the first and third fluids) removedfrom it that mixes with the fluid in the fifth fluid plug 10-5. Thefifth fluid plug 10-5 may subsequently have a fluid portion 10-5Dremoved from it that contains a mixture of the first, third, and fifthfluids. This fluid portion may mix with seventh fluid plug 10-7.

In some embodiments, the mixing and process of removal of a fluid from afluid plug may continue until each fluid plug contains fluid from atleast a portion of the miscible fluids upstream. However, in otherembodiments, only fluids from certain fluid plugs are mixed with oneanother, while fluids from other fluids plugs are not mixed. The amountof mixing and the number of fluids plugs that are mixed together may becontrolled, for example, by determining the length of intervening fluidsbetween fluid plugs, the volume of the fluid plugs, the phase of thefluid plugs, the viscosity of the fluid plugs, the flow speed of thefluid plugs, the surface tension of the fluids, the polarity of thefluids, the density of the fluids, the interfacial surface tensionbetween adjacent fluids, interfacial surface tension between the fluidplug and the channel wall, channel design (e.g., geometry, length,radius of curvature of corners), and properties of the channel wall(e.g., surface roughness, surface texture, surface energy). Otherfactors may also contribute to the amount of mixing.

It should also be appreciated that while removal of a fluid portion froma fluid plug may result in that fluid portion being added to anotherfluid plug (which results in mixing) in some embodiments, in otherembodiments, the fluid portion is not added to another fluid plug anddoes not result in mixing between fluid plugs. The fluid plug may beused for a different purpose, such as for priming the walls of thechannel segment (e.g., to change the surface tension of the channelwall) or for other purposes. For example, in some embodiments, as afluid plug (e.g., first fluid plug 10-1 of FIG. 3A) flows in the channelsegment, fluid portion 10-1B is removed from the fluid plug butcontinues to travel down the channel segment from a downstream side toan upstream side. The fluid may be designed to not substantially mixwith any subsequent fluid in the channel segment and may end up in thewaste region without being substantially combined into a fluid plug.Other configurations of fluid flow are also possible.

In some embodiments, the amount of mixing and/or the number of fluidsplugs that are mixed together may be controlled by certaincharacteristics of the fluid plugs. In some embodiments, the amountand/or duration of mixing may be controlled in part by the distancebetween fluid plugs or the length/volume of the intervening fluids in achannel segment. For example, if it is desirable to have two fluids mix,they may be positioned relatively close to one another in a channelsegment (e.g., first and third fluids in FIG. 1A). If it is undesirableto have two fluids mix, they may be positioned relatively farther awayfrom one another in a channel segment (e.g., first and eleventh fluidsin FIG. 1A). In certain embodiments, a longer (more volumous)intervening fluid plug will separate fluid plugs to a greater extentthan a shorter (less volumous) intervening fluid plug, and may preventtwo fluid plugs from mixing due to their long separation in the channelsegment. In some instances, a larger percentage of volume reduction of afluid plug, for a given channel length and flow time, may be achievedwith a shorter (less volumous) fluid plug compared to a longer (morevolumous) fluid plug.

The phase of the fluid plugs may be used, in some instances, to preventmixing. For instance, a fluid plug in the liquid phase and its liquidfluid portion may not be able to mix with fluid plugs in the gas phase.Accordingly, where it is desirable to have fluids mix, such fluids maybe miscible with one another to facilitate mixing in some embodiments.Where it is undesirable to have fluids mix, they may be designed to beimmiscible with one another in certain embodiments.

In some cases, the viscosity of the fluid plug may influence mixingwithin the fluid plug. For example, a more viscous fluid plug may havereduced mixing through various mechanisms, such as circulating currentsand diffusion, compared to a less viscous fluid plug. A relatively moreviscous fluid may also deposit less fluid on the walls of a channelsegment during fluid flow compared to a relatively less viscous fluid insome embodiments.

The flow speed of the fluid plugs may also influence mixing within afluid plug and the removal of a fluid portion from the fluid plug (e.g.,deposition of the fluid portion on a wall of the channel segment). Forinstance, faster flow speeds may result in larger amounts of fluid beingremoved from a fluid plug, for a given amount of flow time, compared toremoval at slower flow speeds. In some embodiments, slower flow speedsmay result in enhanced diffusion of a fluid portion into a fluid plugcompared to flow at higher flow speeds.

In some instances, mixing may be controlled using more than onecharacteristic, such as more than one of the characteristics describedabove (e.g., volume and phase of the fluids). Other methods ofcontrolling mixing based on characteristics of the fluid plugs are alsopossible. In certain embodiments, the amount of mixing and/or the numberof fluids plugs that are mixed together may be controlled by certainproperties of the fluids. For instance, a fluid or fluid plug that has alower surface tension with respect to a channel wall may more readilyfacilitate removal of a fluid portion from the fluid plug (e.g., producea fluid portion that is deposited on the channel wall) than afluid/fluid plug that has a higher surface tension with respect to thechannel wall. Thus, the relative surface tension of the fluid can bevaried to control the amount of fluid removed from a fluid plug (and,therefore, the subsequent amount of mixing between fluids).

In certain embodiments, the surface tension between a fluid and achannel wall may be selected as desired. In some cases, a wetting agentmay be added to a fluid or fluid plug to control the surface tension.The wetting agent may be added, for example, prior to mixing, as aresult of mixing, or as a result of a fluid being removed from a fluidplug. In certain cases, a wetting agent may be added to the channel wallto control surface tension, e.g., during manufacturing of the device,prior to fluid flow, and/or as a result of fluid flow. In general, anysuitable wetting agent at any desired concentration may be used.Examples of suitable wetting agents include, but are not limited to,polyvinyl alcohol, non-ionic detergents (e.g., poly(ethylene oxide)derivatives like Tween 20 and Triton, fatty alcohols), anionicdetergents (e.g., sodium dodecyl sulfate and related detergents withshorter or longer alkane chains such as sodium decyl sulfate or sodiumoctadecyl sulfate, or fatty acid salts), cationic detergents (e.g.,quaternary ammonium cations such as cetyl trimethylammonium bromide),zwitterionic detergents (e.g., dodecyl betaine), perfluorodetergents(e.g., Capstone FS-10), low surface tension liquids (e.g., alcohols suchas isopropanol), and combinations thereof. In certain embodiments, anon-wetting agent (e.g., ionic compounds) may be added to increase thesurface tension.

In embodiments in which a wetting agent is added to a fluid or fluidplug, the percentage (by weight/volume) of the wetting agent in thefluid or fluid plug may be greater than or equal to about 0.001%,greater than or equal to about 0.01%, greater than or equal to about0.025%, greater than or equal to about 0.05%, greater than or equal toabout 0.1%, greater than or equal to about 0.1%, greater than or equalto about 0.5%, greater than or equal to about 1%, greater than or equalto about 5%, greater than or equal to about 10%, greater than or equalto about 20%, greater than or equal to about 30%, greater than or equalto about 40%, or greater than or equal to about 40%. In some instances,the percentage of wetting agent in the fluid or fluid plug may be lessthan or equal to about 75%, less than or equal to about 50%, less thanor equal to about 40%, less than or equal to about 30%, less than orequal to about 20%, less than or equal to about 10%, less than or equalto about 5%, less than or equal to about 1%, less than or equal to about0.5%, less than or equal to about 0.01%, or less than or equal to about0.01%. Combinations of the above-referenced ranges are also possible(e.g., greater than or equal to about 0.01% or less than or equal toabout 50%). Other ranges of wetting agent percentages are also possible.

Polarity of the fluids may also influence mixing. For example, in someembodiments fluids with differing polarities (e.g., a water based fluidand an oil based fluid) may not mix or may mix to a relatively lesserextent, while fluids with similar polarities (e.g., a water based fluidand a methanol based fluid) may mix or may mix to a relatively greaterextent. In some cases, polarity may be used to prevent or limit adjacentfluids from mixing and/or prevent or limit fluid portions from mixingwith certain non-adjacent fluid plugs. In other cases, polarity may beused to prevent or limit adjacent fluids from mixing and allow fluidportions to mix with certain non-adjacent fluid plugs.

In some instances, the density of the fluids may be used to controlmixing. Significant differences in density between fluids may prevent orlimit the fluids from mixing. Conversely, fluids with similar densitiesmay readily mix.

In certain cases, the interfacial tension between fluids may alsoinfluence mixing. For instance, fluids with a high interfacial tensionwith each other may not mix or may mix to a lesser extent, while fluidswith a low interfacial tension with one another may mix to a relativelygreater extent. In some cases, interfacial tension may be used toprevent or limit adjacent fluids from mixing and prevent or limit fluidportions from mixing with certain non-adjacent fluid plugs. In othercases, interfacial tension may be used to prevent or limit adjacentfluids from mixing and allow fluid portions to mix with certainnon-adjacent fluid plugs.

In some instances, mixing may be controlled using more than one propertydescribed herein (e.g., surface tension and polarity). Other methods ofcontrolling mixing based on properties of the fluids are also possible.

In some embodiments, the amount of mixing and/or the number of fluidsplugs that are mixed together may be controlled by certaincharacteristics of the channel segment. For instance, the geometry ofthe channel segment may be used to control mixing. Non-limiting examplesof geometrical channel features that may influence mixing includecross-sectional shape, cross-sectional area, aspect ratio, hydraulicdiameter, radius of curvature of internal corners, deviations in thechannel (e.g., turns, bends), radius of curvature of deviations in thechannel, and gradual and/or abrupt changes in channel geometry (e.g.,changes in cross-section area). For instance, a channel cross-sectionwith sharper corners may more readily facilitate removal of a fluid froma fluid plug compared to a channel cross-section with blunt corners. Inone example, a channel with a cross-section that includes a radius ofcurvature substantially smaller than the half-width and/or half-heightof the channel may more readily facilitate removal of a fluid from afluid plug compared to a channel cross-section that does not includesuch a radius of curvature, or a channel cross-section having arelatively larger radius of curvature. A radius of curvaturesubstantially smaller than the half-width and/or half-height of thechannel may be, for example, less than or equal to about 50%, less thanor equal to about 40%, less than or equal to about 30%, less than orequal to about 20%, less than or equal to about 10%, or less than orequal to about 5% of the half-width and/or half-height of the channel.Additional examples of channel configurations and dimensions areprovided in more detail below.

The length of the channel segment may also be used to control mixing.For example, longer channel segments may allow greater volume reductionof a fluid plug compared to a shorter channel, with all other factorsbeing equal. In some cases, a channel that is substantially longer thanthe length occupied by the fluid plug may allow greater volume reductionof the fluid (e.g., the entire volume) than a channel that is notsubstantially longer than the length occupied by the fluid plug.Examples of values of lengths are provided in more detail below. In someinstances, mixing may be controlled using more than one characteristic(e.g., cross-section shape and length). Other methods of controllingmixing based on characteristics of the channel are also possible.

In some embodiments, the amount of mixing and/or the number of fluidsplugs that are mixed together may be controlled by certaincharacteristics of a channel wall (e.g., surface roughness, surfacetexture, surface energy, surface polarity, surface charge, interfacialsurface tension between the channel wall and a fluid, local variationsin the characteristics of the channel wall). For instance, the surfaceroughness of a channel wall may be selected to facilitate or preventremoval of a fluid portion from a fluid plug. A channel wall with ahigher surface roughness may more readily facilitate removal of a fluidportion from a fluid plug than a channel wall with a lower surfaceroughness.

In some embodiments, a channel segment (or a portion thereof) may have aroot mean square surface (RMS) roughness of less than about less than orequal to about 10 microns. In certain embodiments, the RMS surfaceroughness may be, for example, less than or equal to about 5 microns,less than or equal to about 3 microns, less than or equal to about 1micron, less than or equal to about 0.8 microns, less than or equal toabout 0.5 microns, less than or equal to about 0.3 microns, less than orequal to about 0.1 microns, less than or equal to about 0.08 microns,less than or equal to about 0.05 microns, less than or equal to about0.08 microns, less than or equal to about 0.01 microns, or less than orequal to about 0.005 microns. In some instances, the RMS surfaceroughness may be greater than or equal to about 0.005 microns, greaterthan or equal to about 0.01 microns, greater than or equal to about 0.05microns, greater than or equal to about 0.1 microns, greater than orequal to about 0.5 microns, greater than or equal to about 1 micron, orgreater than or equal to about 3 microns. Combinations of theabove-referenced ranges are also possible (e.g., greater than or equalto about 0.05 microns and less than or equal to about 5 microns. RMSsurface roughness is a term known to those skilled in the art, and maybe expressed as:

$\sigma_{h} = {\lbrack \langle ( {z - z_{m}} )^{2} \rangle \rbrack^{1/2} = \lbrack {\frac{1}{A}{\int_{A}{( {z - z_{m}} )^{2}\ {\mathbb{d}A}}}} \rbrack^{1/2}}$

where A is the surface to be examined, and |z−z_(m)| is the local heightdeviation from the mean.

In general, surface roughness and/or surface texture of the channel maybe formed during fabrication or later modified using any suitablemethod. Exemplary methods of fabricating or modifying the surfaceroughness and/or surface texture of the channel include chemical etching(e.g., acid, alkaline, corrosive solvent), plasma etching (e.g., lowpressure, atmospheric, flame, plasma etching with inert and/or reactivegases), electrochemical etching, corona discharge, mechanical methods(e.g., mechanical machining, laser machining, mechanical polishing,mechanical grinding, bead-blasting, grit-blasting, shot-peening),ultrasonic machining, electrical methods (e.g., electrochemicalpolishing, electric discharge machining, electroforming), coating (e.g.,by spray-coating, physical vapor deposition, chemical vapor deposition,painting), and combinations thereof. In some instances, the surfaceroughness and/or texture may be produced using a molding process. Thesurface texture and/or roughness of the mold may be modified using anyof the above methods and/or coating or plating the mold surface. Othermethods of producing a desired surface texture and/or surface roughnessare also possible.

In some instances, the surface charge of a channel wall may be used tocontrol mixing. In one example, the surface charge of a channel wall maybe used to facilitate the formation of a fluid portion of an oppositelycharged fluid. In some embodiments, the surface charge density on achannel wall or a portion thereof may be greater than or equal to about0 C/m², greater than or equal to about 0.01 C/m², greater than or equalto about 0.05 C/m², greater than or equal to about 0.1 C/m², or greaterthan or equal to about 0.5 C/m². In some instances, the surface chargedensity on a channel wall or portion thereof may be less than or equalto about 1 C/m², less than or equal to about 0.5 C/m², less than orequal to about 0.1 C/m², or less than or equal to about 0.05 C/m².Combinations of the above-referenced ranges are also possible (e.g.,greater than or equal to about 0 C/m² and less than or equal to about 1C/m²). Other values of surface charge density are also possible.

In some instances, mixing may be controlled using more than onecharacteristic (e.g., surface energy, surface polarity, and surfaceroughness). Other methods of controlling mixing based on characteristicsof a channel wall are also possible. It should also be understood thatother characteristics of a channel wall can be used to control mixing.

In some embodiments, the surface energy of a channel wall or a portionthereof may be used to control mixing. In some instances, the surfaceenergy of a channel wall may be greater than or equal to about 10dynes/cm, greater than or equal to about 25 dynes/cm, greater than orequal to about 50 dynes/cm, greater than or equal to about 75 dynes/cm,greater than or equal to about 100 dynes/cm, greater than or equal toabout 200 dynes/cm, greater than or equal to about 300 dynes/cm, orgreater than or equal to about 400 dynes/cm. In some embodiments, thesurface energy of a channel wall may be less than or equal to about 500dynes/cm, less than or equal to about 400 dynes/cm, less than or equalto about 300 dynes/cm, less than or equal to about 200 dynes/cm, lessthan or equal to about 100 dynes/cm, less than or equal to about 75dynes/cm, or less than or equal to about 25 dynes/cm. Combinations ofthe above-referenced ranges are also possible (e.g., greater than orequal to about 10 dynes/cm and less than or equal to about 200dynes/cm). Other values of surface energy are also possible.

As known to those of ordinary skill in the art, surface energy includesboth a polar component and a dispersion (non-polar) component. In someembodiments, the surface polarity (e.g., as indicated by the ratio ofthe polar component to the dispersive component of the surface energy)of a channel wall or a portion thereof may be used to control mixing.For example, for a cyclo-olefin copolymer the surface polarity is 0(entirely dispersive), for water the surface polarity is 2.3 (fairlypolar), and for plasma-treated surfaces the surface polarity may have aratio of 3 or more.

In some instances, the ratio of the polar component to the dispersivecomponent of the surface energy may be greater than or equal to about 0,greater than or equal to about 0.5, greater than or equal to about 1,greater than or equal to about 1.5, greater than or equal to about 2greater than or equal to about 2.5, greater than or equal to about 3,greater than or equal to about 3.5, or greater than or equal to about 4.In some embodiments, the ratio of the polar component to the dispersivecomponent of the surface energy may be less than or equal to about 5,less than or equal to about 4.5, less than or equal to about 4, lessthan or equal to about 3.5, less than or equal to about 3, less than orequal to about 2.5, less than or equal to about 2, less than or equal toabout 1.5, or less than or equal to about 1. Combinations of theabove-referenced ranges are also possible (e.g., greater than or equalto about 0 and less than or equal to about 3). Other values of surfacepolarity are also possible.

In some embodiments, the surface charge, surface energy, and/or surfacepolarity of the channel may be selected as desired. In general, surfacecharge, surface energy, and/or surface polarity of the channel may beformed during fabrication or later modified using any suitable method.Exemplary methods of fabricating or modifying the surface charge,surface energy, and/or surface polarity of the channel include exposureto reactive agents (e.g., redox agents, permanganate, peroxides, chromicacid, other acids, alkaline solutions, corrosive solvent), plasmaexposure (e.g., low pressure, atmospheric, flame, plasma etching withinert and/or reactive gases), surface functionalization, coating methods(e.g., evaporation, sputtering, vapor deposition processes, electrolessplating, chemical deposition processes, electrochemical depositionprocesses), and combinations thereof. In some instances, a portion ofthe channel may be coated with materials such as metallic material,non-metallic material, nanoparticles, surface reactive agents, aminereactive group (e.g., NHS-activated molecules, molecules with carboxylicacid or aldehyde), thiol-reactive groups (e.g., maleimido-activatedmolecules), carboxy-reactive groups (e.g., amines), polyelectrolyte(e.g., polyethylene amine, dextran sulfate, copolymer with charged sidechains), hydrophobic or partially hydrophobic material (e.g., co-polymerwith hydrophobic chains such as polystyrene), silane (e.g.,methoxysilanes, ethoxysilanes,trichloro(1H,1H,2H,2H-perfluorooctyl)silane, epoxy silanes), parylene,silicon dioxide, polyvinyl pyrrolidone, carbon-based nanostructures(e.g., carbon nanotubes), photosensitive molecules (e.g., derivatives ofdiazirine), biomolecules (e.g., proteins, DNA, carbohydrates, lipids,amino acid side chains), and combinations thereof. Other methods ofproducing a desired surface charge, surface energy, and/or surfacepolarity on channel are also possible.

In certain cases, as shown in illustratively FIG. 3D the entire volumeof a fluid (e.g., the first fluid) may be incorporated into one or morefluid plugs downstream such that the fluid plug is no longer present inthe channel segment. In some cases, the volume of the fluid in the fluidplug may be reduced by a certain percentage (e.g., compared to theinitial volume of the fluid plug). For instance, in some embodiments,the volume of a fluid plug may be reduced by greater than or equal toabout 50%, greater than or equal to about 60%, greater than or equal toabout 70%, greater than or equal to about 80%, greater than or equal toabout 90%, or greater than or equal to about 95%. In some instances, thevolume of a fluid in a fluid plug may be reduced by less than or equalto about 100%, less than or equal to about 90%, less than or equal toabout 80%, less than or equal to about 70%, or less than or equal toabout 60%. Combinations of the above-referenced ranges are also possible(e.g., greater than or equal to about 50% and less than or equal toabout 100%). In some cases, 100% of the volume of the fluid is removedfrom a fluid plug, such that the fluid plug no longer remains in thesystem. In such embodiments, the fluid removed from the fluid plug maybe entirely dispersed along or within the channel. In other embodiments,0% of the fluid is removed from a fluid plug during fluid flow. Othervalues of volume reduction percentage are also possible. As describedherein, in some embodiments the volume of more than one fluid plugs isreduced by the amounts noted above.

In addition to fluid plugs, a channel segment may also contain at leastone substantially dry reagent in some embodiments (e.g., during storageand/or prior to a flowing step described herein). An example of mixingbetween a fluid from a fluid plug and a substantially dry reagent isshown in FIGS. 4A-E. As shown illustratively in FIG. 4A, a channelsegment 5, including an upstream portion 7 and a downstream portion 8,may contain a substantially dry reagent 30, first fluid plug 10-1, asecond fluid plug 20-2, a third fluid plug 10-3. The fluid plugs maycontain a first fluid 10-1A, a second fluid 20-2A, and a third fluid10-3A, respectively. As shown illustratively in this figure, the secondfluid plug may be positioned between the first and third fluid plugs. Insome cases, the second fluid may be immiscible with the first and thirdfluids, while the first and third fluids may optionally be miscible withone another. Additionally, as shown in the figure, the substantially dryreagent may be positioned downstream of the fluid plugs. In general,however, the substantially dry reagent may have any suitable positionrelative to the fluid plugs. For instance, the substantially dry reagentmay be positioned between two fluid plugs in some embodiments. In somecases, a substantially dry reagent is positioned in a gaseous fluid plug(e.g., air) which is flanked on both ends by two liquid fluid plugs.Such a configuration may be appropriate for storage of the reagents incertain embodiments.

As shown in FIG. 4B, the fluids may be flowed in series toward thesubstantially dry reagent, e.g., from upstream to downstream in thedirection of arrow 9. In some embodiments, flowing first fluid plug 10-1over the substantially dry reagent may cause the first fluid to mix withthe reagent (which is no longer substantially dry). The reagent may mixwith the first fluid to form a homogenous or heterogeneous (e.g.,solution or suspension) mixture. During flow, a mixture 10-1C of thefirst fluid and reagent may leave a fluid portion 10-1D, which may beimmiscible with second fluid 20-2A and miscible with third fluid 10-3A,as illustrated in FIG. 3C. As shown in FIGS. 3D-E, fluid portion 10-1Dmay mix with the third fluid in the third fluid plug to form a mixtureof the first fluid, the reagent, and third fluid 10-3E. In certaincases, the entire volume of the mixture of the first fluid and thereagent may be removed from first fluid plug 10-1 and may mix with thethird fluid plug.

Fluids can be flowed in a device described herein using any suitablemethod. In some embodiments, a fluidic device employs one or more ventvalves to controllably flow and/or mix portions of fluid within thesystem. The vent valves can comprise, for example, a port in fluidcommunication with the channel in which a fluid is positioned, and maybe actuated by positioning a seal over the port opening or by removingthe seal from the port opening. In certain embodiments, the seal mayinclude a valving mechanism such as a mechanical valve operativelyassociated with a tube in fluid communication with the port. Generally,opening the vent valve allows the port to function as a vent. When theport functions as a vent, the fluid located on one side of the ventvalve flows, while the fluid located on the opposite side of the ventvalve relative to the first fluid remains stationary. When the valve isclosed, the port no longer functions as a vent, and the fluid located onboth sides of the vent valve can flow through the system towards anoutlet. Advantageously, fluid control such as a sequence of fluid flowand/or a change in flow rate, can be achieved by opening and closing oneor more vent valves and by applying a single source of fluid flow (e.g.,a vacuum) operated at a substantially constant pressure. This cansimplify the operation and use of the device by an intended user. Ventvalves are described in more detail in U.S. Patent Publication No.2011/0120562, filed Nov. 24, 2010 and entitled “Fluid Mixing andDelivery in Microfluidic Systems,” which is incorporated herein byreference in its entirety for all purposes.

In some embodiments, when the fluid flow source is activated, one ormore channels in the fluidic device may be pressurized (e.g., toapproximately −30 kPa) which may drive the fluids within the channeltoward the outlet. In some embodiments, fluids can be stored serially ina channel upstream of a vent valve positioned along the channel, andafter closing the vent valve, the fluids can flow sequentially towardsthe channel outlet. In some cases, fluids can be stored in separate,intersecting channels, and after closing a vent valve the fluids can beflowed sequentially. The timing of delivery and the volume of fluid canbe controlled, for example, by the timing of the vent valve actuation.

An example of controlling movement of fluid plugs in a fluidic devicecomprising multiple channel segments (e.g., branching channels) and atleast one vent valve is shown in FIGS. 6A-6C. In the device illustratedin FIG. 6A, a channel segment 210 is fluidically connected to twochannel segments (e.g., branching channels) 212 and 214, whichintersected at vent valve 216. As shown in this figure, channel segment210 may optionally contain fluid plug 218. In some embodiments, fluidsplugs 220 and 222 may be stored and/or sealed in channel segments 212and 214, respectively (e.g., prior to first use of the device). Channelsegment 210 is shown connected to outlet 224, while channel segments 212and 214 are shown connected to inlets 226 and 228, respectively. All ofthe fluids in the device may be separated by plugs of gas (immisciblewith fluid plugs 218, 220 and 222).

As shown illustratively in FIG. 6B, fluids 220 and 222 may betransported sequentially. To transport fluid plug 222, vent valve 216and inlet 228 may be both closed (while inlet 226 is opened). Totransport fluid plug 220 after fluid plug 222 is transported, vent valve216 and inlet 226 may be both closed (while inlet 228 is opened). Mixingcan then occur between fluid plugs 218, 222 and/or 220 in channelsegment 210 as described herein (e.g., with respect to FIGS. 1-4). Thetiming of when the vent valves are opened or closed can be used to varythe length/volume of the plugs of gas separating fluid plugs 218, 222and/or 220, as well as the duration of fluid flow.

Advantageously, vent valves can be operated without constricting thecross-section of the microfluidic channel on which they operate, asmight occur with certain valves in the prior art. Such a mode ofoperation can be effective in preventing leaking across the valve.Moreover, because vent valves can be used, some systems and methodsdescribed herein do not require the use of certain internal valves,which can be problematic due to, for example, their high expense,complexity in fabrication, fragility, limited compatibility with mixedgas and liquid systems, and/or unreliability in microfluidic systems.

It should be understood that while vent valves are described, othertypes of valving mechanisms can be used with the systems and methodsdescribed herein. Non-limiting examples of a valving mechanism which maybe operatively associated with a valve include a diaphragm valve, ballvalve, gate valve, butterfly valve, globe valve, needle valve, pinchvalve, poppet valve, or pinch valve. The valving mechanism may beactuated by any suitable means, including a solenoid, a motor, by hand,by electronic actuation, or by hydraulic/pneumatic pressure.

As described herein, in some embodiments, reagents (e.g., for a chemicaland/or biological reaction) may be stored in fluid and/or dry form in afluidic device. The method of storage may depend on the particularapplication. Reagents can be stored, for example, as a liquid, a gas, agel, a plurality of particles, or a film. The reagents may be positionedin any suitable portion of a device, including, but not limited to, in achannel or channel segment, reservoir, on a surface, and in or on amembrane, which may be part of a reagent storage area. A reagent may beassociated with a fluidic system (or components of a system) in anysuitable manner. For example, reagents may be crosslinked (e.g.,covalently or ionically), absorbed, or adsorbed (physisorbed) onto asurface within the fluidic system. In some cases, a liquid is containedwithin a channel or reservoir of a device.

In certain embodiments, one or more channel segments of a fluidic deviceincludes a stored liquid reagent (e.g., in the form of a fluid plug). Insome cases, more than one liquid reagents (e.g., fluid plugs) are storedin a channel or channel segment. The liquid reagents may be separated bya separation fluid, which may be immiscible with the liquid reagents.The fluid reagents may be stored in the device prior to first use, orintroduced into the device at first use. In some cases, the liquidreagents may be kept separate during storage of the fluids (e.g., whilethe device is sealed). During use of the device, at least portions ofthe liquids may be combined (e.g., mixed) using the methods describedherein.

Certain fluidic devices may be designed to include both liquid and dryreagents stored in a single article prior to first use and/or prior tointroduction of a sample into the device. In some cases, the liquid anddry reagents are stored in fluid communication with each other prior tofirst use. In other cases, the liquid and dry reagents are not in fluidcommunication with one another prior to first use, but at first use areplaced in fluid communication with one another. For instance, one ormore liquid reagents may be stored in a first common channel and one ormore dry reagents stored in a second common channel, the first andsecond common channels not being connected or in fluidic communicationwith one another prior to first use. Additionally or alternatively, thereagents may be stored in separate vessels such that a reagent is not influid communication with the fluidic device prior to first use. The useof stored reagents can simplify use of the fluidic device by a user,since this minimizes the number of steps the user has to perform inorder to operate the device. This simplicity can allow the fluidicdevices described herein to be used by untrained users, such as those inpoint-of-care settings, and in particular, for devices designed toperform immunoassays.

In various embodiments involving the storage of fluid (e.g., liquid)reagents prior to first use, the fluids may be stored (and, in someembodiments, statically maintained without mixing) in a fluidic devicefor greater than 10 seconds, one minute, one hour, one day, one week,one month, or one year. By preventing contact between certain fluids,fluids containing components that would typically react or bind witheach other can be prevented from doing so, e.g., while being maintainedin a common channel. For example, while they are stored, fluids (e.g.,in the form of fluid plugs) may be kept separated at least in part byimmiscible separation fluids so that fluids that would normally reactwith each other when in contact may be stored for extended periods oftime in a common channel. In some embodiments, the fluids may be storedso that they are statically maintained and do not move in relation totheir position in the channel. Even though fluids may shift slightly orvibrate and expand and contract while being statically maintained,certain fluidic devices described herein are adapted and arranged suchthat fluids in a common channel do not mix with one another during theseprocesses.

Fluidic devices that are used for storage of one or more reagents (e.g.,prior to first use) may be stored at reduced temperatures, such as lessthan or equal to 10° C., 4° C., 0° C., or −10° C. Fluids may also beexposed to elevated temperatures such as greater than 25° C., greaterthan 35° C. or greater than 50° C. Fluids may be shipped from onelocation to the other by surface or air without allowing for mixing ofreagent fluids contained in the channel. The amount of separation fluidmay be chosen based on the end process with which the fluids are to beused as well as on the conditions to which it is expected that thefluidic device will be exposed. For example, if the fluidic device isexpected to receive physical shock or vibration, fluids may only fillportions but not all of a channel segment. Furthermore, larger plugs ofimmiscible separation fluid may be used along with one or more channelconfigurations described herein. In this manner, distinct fluids withina channel system of a fluidic device may avoid mixing.

A fluidic device may include one or more characteristics that facilitatecontrol over fluid transport and/or prevent fluids from mixing with oneanother during storage. For example, a device may include structuralcharacteristics (e.g., an elongated indentation or protrusion) and/orphysical or chemical characteristics (e.g., hydrophobicity vs.hydrophilicity) or other characteristics that can exert a force (e.g., acontaining force) on a fluid. In some cases, a fluid may be held withina channel using surface tension (e.g., a concave or convex meniscus).For example, certain portions of a channel segment may be patterned withhydrophobic and hydrophilic portions to prevent movement and/or mixingof fluids during storage. One measure of hydrophobicity that can beuseful in selecting such materials is contact angle measurements takenbetween water and a candidate material. While “hydrophobic” can beconsidered a relative term in some cases, a particular degree or amountof hydrophobicity can be easily selected by those of ordinary skill inthe art, with the aid of knowledge of the characteristics of particularmaterials and/or readily-determined contact angle measurements forselecting fluids and/or materials described herein.

In some cases, a channel may segment have an absence of inner walls orother dividers to keep the fluids apart and fluids may be separated by aseparation fluid as described herein.

In some embodiments, fluids can be stored on two sides of a fluidicdevice, as described in more detail in U.S. Patent Publication No.2010/0158756, filed Dec. 17, 2009, entitled “Reagent Storage inMicrofluidic Systems and Related Articles and Methods,” which isincorporated herein by reference in its entirety for all purposes. Insome cases, the fluidic device may include channel segments havingnon-circular cross sections and channel segments having circularcross-sections. In certain embodiments, at least some of the channelsegments having circular cross-sections may pass through the thicknessof the article and may connect channels formed on either surfaces of thearticle.

In some embodiments, a channel segment may include one or more corners(e.g., curved corners) having a certain radius of curvature. The curvedcorner may be, for example, a convex portion of a surface that mateswith a cover. The convex portion of the surface may be formed duringfabrication of the channel segment by various techniques (e.g.,injection molding). In certain embodiments, a channel segment mayinclude one or more corners (e.g., curved corners) having a radius ofcurvature of, for example, less than or equal to about 100 μm, less thanor equal to about 50 μm, less than or equal to about 30 μm, less than orequal to about 20 μm, less than or equal to about 10 μm, less than orequal to about 5 μm, less than or equal to about 3 μm, less than orequal to about 2 μm, less than or equal to about 1 μm, less than orequal to about 0.5 μm, or less than or equal to about 0.1 μm. In someembodiments, the radius of curvature of a curved corner of a channel maybe, e.g., greater than or equal to about 0.1 μm, greater than or equalto about 0.5 μm, greater than or equal to about 1 μm, greater than orequal to about 2 μm, greater than or equal to about 3 μm, greater thanor equal to about 5 μm, greater than or equal to about 10 μm, greaterthan or equal to about 20 μm, greater than or equal to about 30 μm,greater than or equal to about 50 μm, or greater than or equal to about100 μm. Combinations of the above-noted ranges are also possible (e.g.,a radius of curvature of greater than or equal to about 1 micron andless than or equal to about 20 microns). Other ranges are also possible.A curved corner having a relatively smaller radius of curvature mayincrease the amount of fluid being removed from a fluid plug flowingalong a portion of the channel, compared to a fluid plug flowing in achannel having a relatively larger radius of curvature.

In some embodiments, a channel having a curved corner may have a ratioof a cross-sectional dimension (e.g., a width or a height) of thechannel to the radius of curvature of the substantially curved corner ofgreater than or equal to about 1:1, greater than or equal to about 2:1,greater than or equal to about 3:1, greater than or equal to about 5:1,greater than or equal to about 10:1, greater than or equal to about20:1, greater than or equal to about 30:1, greater than or equal toabout 50:1, greater than or equal to about 100:1, greater than or equalto about 200:1, or greater than or equal to about 500:1. In someinstances, the ratio of a cross-sectional dimension (e.g., a width or aheight) of the channel to the radius of curvature of the substantiallycurved corner may be less than or equal to about 600:1, less than orequal to about 400:1, less than or equal to about 200:1, less than orequal to about 100:1, less than or equal to about 75:1, less than orequal to about 50:1, less than or equal to about 25:1, less than orequal to about 10:1, or less than or equal to about 5:1. Combinations ofthe above-referenced ranges are also possible (e.g., greater than orequal to about 5:1 and less than or equal to about 400:1). Other valuesof the ratio of a cross-sectional dimension (e.g., a width or a height)of the channel to the radius of curvature of the substantially curvedcorner are also possible.

In some fluidic devices described herein, it is desirable to havefluidic components (e.g., channel, channel segment, channel portion)having non-zero draft angles. As known to those of ordinary skill in theart, a draft angle is the amount of taper, e.g., for molded or castparts, perpendicular to the parting line. For example, as shownillustratively in FIG. 5A, a substantially rectangular channel 125,which has walls 125-A and 125-C that are substantially perpendicular tosurface 121 (e.g., a parting line), has a draft angle 196 of 0°. Thecross sections of fluidic channels having non-zero draft angles, on theother hand, may resemble a trapezoid, a parallelogram, or a triangle.For example, as shown in the embodiment illustrated in FIG. 5B, channel127 has a substantially trapezoidal cross-section. Draft angle 196 isformed by the angle between a line perpendicular to surface 121 and wall127-A of the channel, and is non-zero in this embodiment.

In some embodiments, during fluid flow a corner of a channel having adraft angle less than 90° may cause a fluid to deposit a relativelylarger fluid portion than a corner of a channel having a draft anglegreater than or equal to 90°, as shown in FIG. 12. FIG. 12A shows across-section of a channel portion including corners a draft angle 200less than 90° and a draft angle 205 greater than 90°. During fluid flow,a fluid portion 201 in the corner of the channel encompassing draftangle 200 may be greater than a fluid portion 206 in the cornerencompassing draft angle 205, as shown illustratively in FIG. 12B. Incertain embodiments, the amount of a fluid portion deposited in a cornerof a channel may increase with decreasing draft angle. For example, morefluid may be deposited in channel portion 215 than channel portion 210shown in FIG. 12C.

The draft angle of a channel, channel segment, or channel portion, forexample, greater than or equal to about to about 1°, greater than orequal to about 2°, greater than or equal to about 3°, greater than orequal to about 5°, greater than or equal to about 8°, greater than orequal to about 10°, greater than or equal to about 20°, greater than orequal to about 30°, greater than or equal to about 45°, greater than orequal to about 60°, or greater than or equal to about 75°. In someinstances, the draft angle may be less than or equal to about 90°, lessthan or equal to about 75°, less than or equal to about 60°, less thanor equal to about 45, less than or equal to about 30°, less than orequal to about 20°, less than or equal to about 10°, or less than orequal to about 5°. Combinations of the above-referenced ranges are alsopossible (e.g., greater than or equal to about 1° and less than or equalto about 60°).

It should be understood that a channel, channel segment, or channelportion can have any suitable cross-sectional dimension, which maydepend on, for example, where the channel is positioned, how the channelis to be used (e.g., for mixing or for storage of reagents), the size ofthe fluidic device, the volume of reagents intended to flow in thedevice, etc. For instance, in some embodiments, a channel, channelsegment, channel portion, etc. may have a maximum cross-sectionaldimension (e.g., a width or height) of less than or equal to about 5 mm,less than or equal to about 3 mm, less than or equal to about 1 mm, lessthan or equal to about 750 microns, less than or equal to about 600microns, less than or equal to about 500 microns, less than or equal toabout 300 microns, less than or equal to about 200 microns, less than orequal to about 100 microns, less than or equal to about 50 microns, lessthan or equal to about 25 microns, less than or equal to about 10microns, or less than or equal to about 5 microns. In some instances, achannel, channel segment, or channel portion, may have a maximumcross-sectional dimension of greater than or equal to about 0.1 microns,greater than or equal to about 1 microns, greater than or equal to about5 microns, greater than or equal to about 10 microns, greater than orequal to about 25 microns, greater than or equal to about 50 microns,greater than or equal to about 100 microns, greater than or equal toabout 200 microns, greater than or equal to about 400 microns, greaterthan or equal to about 600 microns, greater than or equal to about 900microns, greater than or equal to about 1 mm, greater than or equal toabout 1.5 mm, or greater than or equal to about 3 mm Combinations of theabove-referenced ranges are also possible (e.g., greater than or equalto about 1 micron and less than or equal to about 1 mm) Other values ofmaximum cross-sectional dimensions are also possible.

In some cases, at least one or at least two cross-sectional dimensions(e.g., a height and a width) of a channel, channel segment, or channelportion may be less than or equal to about 2 mm, less than or equal toabout 1 mm, less than or equal to about 750 microns, less than or equalto about 500 microns, less than or equal to about 300 microns, less thanor equal to about 200 microns, less than or equal to about 100 microns,less than or equal to about 50 microns, less than or equal to about 25microns, less than or equal to about 10 microns, or less than or equalto about 5 microns. In some instances, at least one or at least twocross-sectional dimensions of a channel, channel segment, channelportion, etc. may be greater than or equal to about 0.1 microns, greaterthan or equal to about 1 micron, greater than or equal to about 5microns, greater than or equal to about 10 microns, greater than orequal to about 25 microns, greater than or equal to about 50 microns,greater than or equal to about 100 microns, greater than or equal toabout 200 microns, greater than or equal to about 400 microns, greaterthan or equal to about 600 microns, or greater than or equal to about700 microns. Combinations of the above-referenced ranges are alsopossible (e.g., greater than or equal to about 10 μm and less than orequal to about 500 μm). Other values are also possible.

A channel, channel segment, or channel portion may have a certainwidth-to-height ratio. In certain instances, the ratio of the width toheight of a channel, channel segment, or channel portion may be greaterthan or equal to about 1:1, greater than or equal to about 2:1, greaterthan or equal to about 5:1, greater than or equal to about 10:1, greaterthan or equal to about 15:1, or greater than or equal to about 20:1. Insome instances the width-to-height ratio may be less than or equal toabout 30:1, less than or equal to about 20:1, less than or equal toabout 15:1, less than or equal to about 10:1, less than or equal toabout 5:1, or less than or equal to about 2:1. Combinations of theabove-referenced ranges are also possible (e.g., greater than or equalto about 1:1 and less than or equal to about 20:1). Other values arealso possible.

A channel, channel segment, or channel portion may also have an aspectratio (length to largest average cross-sectional dimension) of at least2:1, more typically at least 3:1, 5:1, or 10:1. In some cases, thechannels, channel segments, or channel portions have very large aspectratios, e.g., at least 100:1, 500:1 or 1000:1. Such long channels may beuseful for mixing large volumes of fluids and/or large numbers ofdifferent fluid plugs in the channel. For instance, the channel, channelsegment, or channel portion may contain greater than or equal to 3, 5,10, 20, 30, or 50 fluid plugs (e.g., the fluid reagents and separatingfluids being counted as different plugs). In certain embodiments, achannel, channel segment, or channel portion has a length to largestwidth of less than or equal to 10, 7, 5, 3, or 2. Short channels may beuseful in certain devices for mixing smaller volumes of fluids.

A channel, channel segment, or channel portion may have a length and/orvolume for mixing as described herein. In some embodiments a channel,channel segment, or channel portion may have a volume of greater than orequal to about 0.001 picoliters, greater than or equal to about 0.01picoliters, greater than or equal to about 0.1 picoliters, greater thanor equal to about 1 picoliters, greater than or equal to about 10picoliters, greater than or equal to about 100 picoliters, greater thanor equal to about 0.001 microliters, greater than or equal to about 0.01microliters, greater than or equal to about 0.1 microliters, greaterthan or equal to about 1 microliter, greater than or equal to about 10microliters, greater than or equal to about 25 microliters, greater thanor equal to about 50 microliters, greater than or equal to about 100microliters, greater than or equal to about 150, or greater than orequal to about 200 microliters. In some instances, a channel, channelsegment, or channel portion may have a volume of less than or equal toabout 250 microliters, less than or equal to about 200 microliters, lessthan or equal to about 150 microliters, less than or equal to about 100microliters, less than or equal to about 50 microliters, less than orequal to about 25 microliters, less than or equal to about 15microliters, less than or equal to about 10 microliters, less than orequal to about 5 microliters, less than or equal to about 1 microliters,less than or equal to about 0.1 microliters, or less than or equal toabout 0.01 microliters, less than or equal to about 0.001 microliter,less than or equal to about 100 picoliter, less than or equal to about10 picoliter, less than or equal to about 1 picoliter, or less than orequal to about 0.1 picoliter, less than or equal to about 0.01picoliter. Combinations of the above-referenced ranges are also possible(e.g., greater than or equal to about 0.001 picoliters and less than orequal to about 200 microliters). Other volumes are also possible.

In some embodiments, a channel, channel segment, or channel portion mayhave a length of greater than or equal to about 1 mm, greater than orequal to about 5 mm, greater than or equal to about 10 mm, greater thanor equal to about 20 mm, greater than or equal to about 40 mm, greaterthan or equal to about 60 mm, or greater than or equal to about 80 mm.In some instances, the length may be less than or equal to about 100 mm,less than or equal to about 90 mm, less than or equal to about 70 mm,less than or equal to about 50 mm, less than or equal to about 30 mm, orless than or equal to about 10 mm Combinations of the above-referencedranges are also possible (e.g., greater than or equal to about 1 mm andless than or equal to about 100 mm) Other values of length are alsopossible.

A channel, channel segment, or channel portion may have any suitableconfiguration. In some embodiments, a channel, channel segment, orchannel portion may be a common channel, a branching channel, a channelsegment on a side of a device that is separated from another channelsegment by an intervening channel (e.g., a channel segment passingthrough the thickness of the device, as part of a two-sided device), orany other suitable configuration. In some cases, channel segments orchannel portions may be separated from one another by a component (e.g.,a vent valve or port), or may differ from one another based on a featureof the channel segment or portion (e.g., surface roughness, dimension,etc.). Other configurations are also possible.

A channel, channel segment, or channel portion can be covered oruncovered. In embodiments where it is covered, at least one portion ofthe channel can have a cross-section that is substantially enclosed, orthe entire channel may be substantially enclosed along its entire lengthwith the exception of its inlet(s) and outlet(s). One or more inlet(s)and/or outlet(s) may also be enclosed and/or sealed. In certainembodiments, one or more covers is adapted and arranged such that achannel segment, an inlet, and/or an outlet is substantially enclosedand/or sealed prior to first use of the device by a user, but opened orunsealed at first use. In some embodiments, such a configuration maysubstantially prevent fluids and/or other reagents stored in the devicefrom being removed from the device (e.g., due to evaporation) duringfabrication, shipping, and/or storage of the device, as describedherein.

As used herein, “prior to first use” of the device means a time or timesbefore the device is first used by an intended user after commercialsale. First use may include any step(s) requiring manipulation of thedevice by a user. For example, first use may involve one or more stepssuch as puncturing a sealed inlet or removing a cover from an inlet tointroduce a reagent into the device, connecting two or more channels tocause fluid communication between the channels, preparation of thedevice (e.g., loading of reagents into the device) before analysis of asample, loading of a sample onto or into the device, preparation of asample in a region of the device, performing a reaction with a sample,detection of a sample, etc. First use, in this context, does not includemanufacture or other preparatory or quality control steps taken by themanufacturer of the device. Those of ordinary skill in the art are wellaware of the meaning of first use in this context, and will be ableeasily to determine whether a device of the invention has or has notexperienced first use. In one set of embodiments, devices of theinvention are disposable after first use, and it is particularly evidentwhen such devices are first used, because it is typically impractical touse the devices at all after first use.

A fluidic device, or portions thereof, can be fabricated of any materialsuitable for forming a channel or other component. Non-limiting examplesof materials include polymers (e.g., polyethylene, polystyrene,polymethylmethacrylate, polycarbonate, poly(dimethylsiloxane), PVC,PTFE, PET, and a cyclo-olefin copolymer), or metals including nickel,copper, stainless steel, bulk metallic glass, or other metals or alloys,or ceramics including glass, quartz, silica, alumina, zirconia, tungstencarbide, silicon carbide, or non-metallic materials such as graphite,silicon, or others. The material forming the fluidic device and anyassociated components (e.g., a cover) may be hard or flexible. Those ofordinary skill in the art can readily select suitable material(s) basedupon e.g., its rigidity, its inertness to (e.g., freedom fromdegradation by) a fluid to be passed through it, its robustness at atemperature at which a particular device is to be used, itstransparency/opacity to electromagnetic waves (e.g., light in theultraviolet and visible regions, terahertz waves, microwaves, and soon), and/or the method used to fabricate features in the material. Forinstance, for injection molded or other extruded articles, the materialused may include a thermoplastic (e.g., polypropylene, polystyrene,polyethylene, polymethylmethacrylate, cyclo-olefin copolymer,polycarbonate, acrylonitrile-butadiene-styrene, nylon 6, PVC, PTFE,PET), an elastomer (e.g., polyisoprene, isobutene-isoprene, nitrile,neoprene, ethylene-propylene, hypalon, silicone), a thermoset (e.g.,epoxy, unsaturated polyesters, phenolics), or combinations thereof. Insome embodiments, fluidic devices including two or more components orlayers may be formed in different materials to tailor the components tothe major function(s) of the each of the components, e.g., based uponthe factors described herein.

In some embodiments, the material and dimensions (e.g., thickness) of afluidic device (and/or cover of a device) are chosen such that it issubstantially impermeable to water vapor. For instance, a fluidic devicedesigned to store one or more fluids therein prior to first use mayinclude a cover comprising a material known to provide a high vaporbarrier, such as metal foil, certain polymers, certain ceramics andcombinations thereof. In other cases, the material is chosen based atleast in part on the shape and/or configuration of the fluidic device.For instance, certain materials can be used to form planar deviceswhereas other materials are more suitable for forming devices that arecurved or irregularly shaped.

In some instances, a fluidic device is comprised of a combination of twoor more materials, such as the ones listed above. For instance, channelsof the fluidic device may be formed in polystyrene or other polymers(e.g., by injection molding) and a biocompatible tape may be used toseal the channels. The biocompatible tape or flexible material mayinclude a material known to improve vapor barrier properties (e.g.,metal foil, polymers or other materials known to have high vaporbarriers), and may optionally allow access to inlets and outlets bypuncturing or unpeeling the tape. A variety of methods can be used toseal a microfluidic channel or portions of a channel, or to joinmultiple layers of a device, including but not limited to, the use ofadhesives, use adhesive tapes, gluing, bonding, welding, brazing,lamination of materials, or by mechanical methods (e.g., clamping,snapping mechanisms, etc.).

In some instances, a fluidic device comprises a combination of two ormore separate components (e.g., layers or fluidic devices) mountedtogether. Independent channel networks, which may optionally includereagents stored and/or sealed therein prior to first use, may beincluded on or in the different components of the fluidic device. Theseparate components may be mounted together or otherwise associated withone another by any suitable means, such as by the methods describedherein, e.g., to form a single (composite) fluidic device. In someembodiments, two or more channel networks are positioned in differentcomponents or layers of the fluidic device and are not connectedfluidically prior to first use, but are connected fluidically at firstuse, e.g., by use of a fluidic connector, as described in more detail inU.S. Pat. No. 8,202,492, issued Jun. 19, 2012 (filed May 1, 2008) andentitled “Fluidic Connectors and Microfluidic Systems.” In otherembodiments, the two or more channel networks are connected fluidicallyprior to first use.

Advantageously, each of the different components or layers that form acomposite fluidic device may be tailored individually depending on thedesigned function(s) of that component or layer. For example, in one setof embodiments, one component of a composite fluidic device may betailored for storing wet reagents. In some such embodiments, thatcomponent may be formed in a material having a relatively low vaporpermeability. Additionally or alternatively, e.g., depending on theamount of fluids to be stored, the storage region(s) of that fluidicdevice may be made with larger cross-sectional dimensions than channelsor regions of other components not used for storage of liquids. Thematerial used to form the fluidic device may be compatible withfabrication techniques suitable for forming larger cross-sectionaldimensions. By contrast, a second component that may be tailored fordetection of an analyte may, in some embodiments, include channelportions having smaller cross-sectional dimensions. Smallercross-sectional dimensions may be useful, for example, in certainembodiments to allow more contact time between fluids flowing in thechannel (e.g., a reagent solution or a wash fluid) and an analyte boundto a surface of the channel, for a given volume of fluid. Additionallyor alternatively, a channel portion of the second component may have alower surface roughness compared to a channel portion of anothercomponent. The smaller-cross sectional dimensions or lower surfaceroughness of the channel portions of the second component may, incertain embodiments, require a certain fabrication technique orfabrication tool different from that used to form a different componentof the fluidic device. Furthermore, in some particular embodiments, thematerial used for the second component may be well characterized forprotein attachment and detection. As such, it may be advantageous toform different channels segments used for different purposes ondifferent components of a fluidic device, which can then be joinedtogether prior to use by an intended user.

Additional characteristics and examples of fluidic devices andcomponents thereof that can be combined with aspects described hereinare described in more detail in U.S. Patent Publication No.2011/0256551, filed Apr. 15, 2011 and entitled “Systems and Devices forAnalysis of Samples,” which is incorporated herein by reference in itsentirety for all purposes.

The methods and systems described herein may involve variety ofdifferent types of analyses, and can be used to determine a variety ofdifferent samples. In some cases, an analysis involves a chemical and/orbiological reaction. In some embodiments, a chemical and/or biologicalreaction involves binding. Different types of binding may take place influidic devices described herein. Binding may involve the interactionbetween a corresponding pair of molecules that exhibit mutual affinityor binding capacity, typically specific or non-specific binding orinteraction, including biochemical, physiological, and/or pharmaceuticalinteractions. Biological binding defines a type of interaction thatoccurs between pairs of molecules including proteins, nucleic acids,glycoproteins, carbohydrates, hormones and the like. Specific examplesinclude antibody/antigen, antibody/hapten, enzyme/substrate,enzyme/inhibitor, enzyme/cofactor, binding protein/substrate, carrierprotein/substrate, lectin/carbohydrate, receptor/hormone,receptor/effector, complementary strands of nucleic acid,protein/nucleic acid repressor/inducer, ligand/cell surface receptor,virus/ligand, etc. Binding may also occur between proteins or othercomponents and cells. In addition, devices described herein may be usedfor other fluid analyses (which may or may not involve binding and/orreactions) such as detection of components, concentration, etc.

In some embodiments, a chemical and/or biological reaction involves areducing agent (e.g., hydroquinone, chlorohydroquinone, pyrogallol,metol, 4-aminophenol and phenidone, Fe(+2), Ti(+3), and V(+2)). In somecases, a chemical and/or biological reaction involves a metal precursor(e.g., a solution of a metal salt, such as a silver salt).

In some cases, a heterogeneous reaction (or assay) may take place in afluidic device; for example, a binding partner may be associated with asurface of a channel, and the complementary binding partner may bepresent in the fluid phase. Other solid-phase assays that involveaffinity reaction between proteins or other biomolecules (e.g., DNA,RNA, carbohydrates), or non-naturally occurring molecules, can also beperformed. Non-limiting examples of typical reactions that can beperformed in a fluidic device include chemical reactions, enzymaticreactions, immuno-based reactions (e.g., antigen-antibody), andcell-based reactions.

Non-limiting examples of analytes that can be determined (e.g.,detected) using fluidic devices described herein include specificproteins, viruses, hormones, drugs, nucleic acids and polysaccharides;specifically antibodies, e.g., IgD, IgG, IgM or IgA immunoglobulins toHTLV-I, HIV, Hepatitis A, B and non A/non B, Rubella, Measles, HumanParvovirus B19, Mumps, Malaria, Chicken Pox or Leukemia; autoantibodies;human and animal hormones, e.g., thyroid stimulating hormone (TSH),thyroxine (T4), vitamin D, vitamin B12, luteinizing hormone (LH),follicle-stimulating hormones (FSH), testosterone, progesterone, humanchorionic gonadotropin, estradiol; other proteins or peptides, e.g.troponin I, troponin T, c-reactive protein, myoglobin, brain natriureticprotein, prostate specific antigen (PSA), free-PSA, complexed-PSA,pro-PSA, EPCA-2, PCADM-1, ABCA5, hK2, beta-MSP (PSP94), AZGP1, AnnexinA3, PSCA, PSMA, JM27, PAP; drugs, e.g., paracetamol or theophylline;marker nucleic acids, e.g., PCA3, TMPRS-ERG; polysaccharides such ascell surface antigens for HLA tissue typing and bacterial cell wallmaterial. Chemicals that may be detected include explosives such as TNT,nerve agents, and environmentally hazardous compounds such aspolychlorinated biphenyls (PCBs), dioxins, hydrocarbons and MTBE.Typical sample fluids include physiological fluids such as human oranimal whole blood, blood serum, blood plasma, semen, tears, urine,sweat, saliva, cerebro-spinal fluid, vaginal secretions; in-vitro fluidsused in research or environmental fluids such as aqueous liquidssuspected of being contaminated by the analyte.

In some embodiments, one or more reagents that can be used to determinean analyte of a sample (e.g., a binding partner of the analyte to bedetermined) is stored and/or sealed in a channel or chamber of a fluidicdevice prior to first use in order to perform a specific test or assay.

In cases where an antigen is being analyzed, a corresponding antibody oraptamer can be the binding partner associated with a surface of amicrofluidic channel. If an antibody is the analyte, then an appropriateantigen or aptamer may be the binding partner associated with thesurface. When a disease condition is being determined, it may bepreferred to put the antigen on the surface and to test for an antibodythat has been produced in the subject. Such antibodies may include, forexample, antibodies to HIV.

In some embodiments, a fluidic device is adapted and arranged to performan analysis involving accumulating an opaque material on a region of achannel segment, exposing the region to light, and determining thetransmission of light through the opaque material. An opaque materialmay include a substance that interferes with the transmittance of lightat one or more wavelengths. An opaque material does not merely refractlight, but reduces the amount of transmission through the material by,for example, absorbing or reflecting light. Different opaque materialsor different amounts of an opaque material may allow transmittance ofless than, for example, 90, 80, 70, 60, 50, 40, 30, 20, 10 or 1 percentof the light illuminating the opaque material. Examples of opaquematerials include molecular layers of metal (e.g., elemental metal),ceramic layers, dyes, polymeric layers, and layers of an opaquesubstance (e.g., a dye). The opaque material may, in some cases, be ametal that can be electrolessly deposited. These metals may include, forexample, silver, gold, copper, nickel, cobalt, palladium, and platinum.Precursors of these metals may be stored and/or flowed in the devicesdescribed herein.

An opaque material that forms in a channel may include a series ofdiscontinuous independent particles that together form an opaque layer,but in one embodiment, is a continuous material that takes on agenerally planar shape. The opaque material may have a dimension (e.g.,a width of length) of, for example, greater than or equal to 1 micron,greater than or equal to 5 microns, greater than 10 microns, greaterthan or equal to 25 microns, or greater than or equal to 50 microns. Insome cases, the opaque material extends across the width of the channel(e.g., a measurement zone) containing the opaque material. The opaquelayer may have a thickness of, for example, less than or equal to 10microns, less than or equal to 5 microns, less than or equal to 1micron, less than or equal to 100 nanometers or less than or equal to 10nanometers. Even at these small thicknesses, a detectable change intransmittance can be obtained. The opaque layer may provide an increasein assay sensitivity when compared to techniques that do not form anopaque layer.

In one set of embodiments, a fluidic device described herein is used forperforming an immunoassay (e.g., for human IgG or PSA) and, optionally,uses silver enhancement for signal amplification. In such animmunoassay, after delivery of a sample (e.g., containing human IgG) toa reaction site or analysis region, binding between two components(e.g., between the human IgG and anti-human IgG) can take place. One ormore reagents, which may be optionally stored in a channel of the deviceprior to use, can then flow over this binding pair complex. Optionally,one of the stored reagents may include a solution of metal colloid(e.g., a gold conjugated antibody) that specifically binds to theantigen to be detected (e.g., human IgG). In other embodiments, themetal colloid can be bound with the sample prior to arriving at thereaction site or analysis region. This metal colloid can provide acatalytic surface for the deposition of an opaque material, such as alayer of metal (e.g., silver), on a surface of the analysis region. Thelayer of metal can be formed by using a two component system: a metalprecursor (e.g., a solution of silver salts) and a reducing agent (e.g.,hydroquinone, chlorohydroquinone, pyrogallol, metol, 4-aminophenol andphenidone, Fe(+2), Ti(+3), and V(+2)), which can optionally be stored indifferent channels prior to use.

Mixing of the two reagents can be performed using the methods describedherein (e.g., as shown illustratively in FIGS. 1-4). In otherembodiments, as a positive or negative pressure differential is appliedto the system, the silver salt and reducing solutions can merge at achannel intersection, where they mix (e.g., due to diffusion) in achannel, and then flow over the analysis region. If antibody-antigenbinding occurs in the analysis region, the flowing of the metalprecursor solution through the region can result in the formation of anopaque layer, such as a silver layer, due to the presence of thecatalytic metal colloid associated with the antibody-antigen complex.The opaque layer may include a substance that interferes with thetransmittance of light at one or more wavelengths. An opaque layer thatis formed in the channel can be detected optically, for example, bymeasuring a reduction in light transmittance through a portion of theanalysis region (e.g., a serpentine channel region) compared to aportion of an area that does not include the antibody or antigen.

Alternatively, a signal can be obtained by measuring the variation oflight transmittance as a function of time, as the film is being formedin an analysis region. The opaque layer may provide an increase in assaysensitivity when compared to techniques that do not form an opaquelayer. Additionally, various amplification chemistries that produceoptical signals (e.g., absorbance, fluorescence, glow or flashchemiluminescence, electrochemiluminescence), electrical signals (e.g.,resistance or conductivity of metal structures created by an electrolessprocess) or magnetic signals (e.g., magnetic beads) can be used to allowdetection of a signal by a detector.

Various types of fluids can be used with the fluidic devices describedherein. As described herein, fluids may be introduced into the fluidicdevice at first use, and/or stored within the fluidic device prior tofirst use. Fluids include liquids such as solvents, solutions andsuspensions. Fluids also include gases and mixtures of gases. The fluidsmay contain any suitable species such as a component for a chemicaland/or biological reaction, a buffer, and/or a detecting agent. Whenmultiple fluids are contained in a fluidic device, the fluids may beseparated by another fluid that is preferably substantially immisciblein each of the first two fluids. For example, if a channel contains twodifferent aqueous solutions, a separation plug of a third fluid may besubstantially immiscible in both of the aqueous solutions. When aqueoussolutions are to be kept separate, substantially immiscible fluids thatcan be used as separators may include gases such as air or nitrogen, orhydrophobic fluids that are substantially immiscible with the aqueousfluids. Fluids may also be chosen based at least in part on the fluid'sreactivity with adjacent fluids, or based on other factors describedherein. For example, an inert gas such as nitrogen may be used in someembodiments and may help preserve and/or stabilize any adjacent fluids.An example of an substantially immiscible liquid for separating aqueoussolutions is perfluorodecalin.

The choice of a separator fluid may be made based on other factors aswell, including any effect that the separator fluid may have on thesurface tension of the adjacent fluid plugs. In some embodiments, it maybe preferred to maximize the surface tension within any fluid plug topromote retention of the fluid plug as a single continuous unit undervarying environmental conditions such as vibration, shock andtemperature variations. Other factors relevant to mixing between fluidsand fluid plugs can also be considered as described herein.

Separator fluids may also be inert to a reaction site (e.g., measurementzone) to which the fluids will be supplied. For example, if a reactionsite includes a biological binding partner, a separator fluid such asair or nitrogen may have little or no effect on the binding partner. Theuse of a gas (e.g., air) as a separator fluid may also provide room forexpansion within a channel of a fluidic device should liquids containedin the device expand or contract due to changes such as temperature(including freezing) or pressure variations.

A variety of determination (e.g., measuring, quantifying, detecting, andqualifying) techniques may be used, e.g., to analyze a sample componentor other component or condition associated with a fluidic describedherein. Determination techniques may include optically-based techniquessuch as light transmission, light absorbance, light scattering, lightreflection and visual techniques. Determination techniques may alsoinclude luminescence techniques such as photoluminescence (e.g.,fluorescence), chemiluminescence, bioluminescence, and/orelectrochemiluminescence. In other embodiments, determination techniquesmay measure conductivity or resistance. As such, an analyzer may beconfigured to include such and other suitable detection systems.

Different optical detection techniques provide a number of options fordetermining reaction (e.g., assay) results. In some embodiments, themeasurement of transmission or absorbance means that light can bedetected at the same wavelength at which it is emitted from a lightsource. Although the light source can be a narrow band source emittingat a single wavelength it may also may be a broad spectrum source,emitting over a range of wavelengths, as many opaque materials caneffectively block a wide range of wavelengths. In some embodiments, asystem may be operated with a minimum of optical devices (e.g., asimplified optical detector). For instance, the determining device maybe free of a photomultiplier, may be free of a wavelength selector suchas a grating, prism or filter, may be free of a device to direct orcollimate light such as a collimator, or may be free of magnifyingoptics (e.g., lenses). Elimination or reduction of these features canresult in a less expensive, more robust device.

Additional examples of detection systems are described in more detailbelow in U.S. Patent Publication No. 2011/0256551, filed Apr. 15, 2011and entitled “Systems and Devices for Analysis of Samples,” which isincorporated herein by reference in its entirety for all purposes.

The articles, components, systems, and methods described herein may becombined with those described in International Patent Publication No.WO2005/066613 (International Patent Application Serial No.PCT/US2004/043585), filed Dec. 20, 2004 and entitled “Assay Device andMethod” [H0498.70211WO00]; International Patent Publication No.WO2005/072858 (International Patent Application Serial No.PCT/US2005/003514), filed Jan. 26, 2005 and entitled “Fluid DeliverySystem and Method” [H0498.70219WO00]; International Patent PublicationNo. WO2006/113727 (International Patent Application Serial No.PCT/US06/14583), filed Apr. 19, 2006 and entitled “Fluidic StructuresIncluding Meandering and Wide Channels” [H0498.70244WO00]; U.S. Pat. No.8,202,492, issued Jun. 19, 2012 (filed May 1, 2008) and entitled“Fluidic Connectors and Microfluidic Systems” [C1256.700000501]; U.S.Patent Publication No. 2009/0075390, filed Aug. 22, 2008, entitled“Liquid Containment for Integrated Assays” [C1256.70001US01]; U.S. Pat.No. 8,222,049, issued Jul. 17, 2012 (filed Apr. 25, 2008), entitled“Flow Control in Microfluidic Systems” [C1256.70002US01]; U.S. Pat. No.8,221,700, issued Jul. 17, 2012 (filed Feb. 2, 2010), entitled“Structures for Controlling Light Interaction with MicrofluidicDevices,” [C1256.70003US01]; U.S. Patent Publication No. 2010/0158756,filed Dec. 17, 2009, entitled “Reagent Storage in Microfluidic Systemsand Related Articles and Methods,” [C1256.70004US01]; U.S. PatentPublication No. 2011/0120562, filed Nov. 24, 2010, entitled “FluidMixing and Delivery in Microfluidic Systems,” [C1256.70005US01]; U.S.Patent Publication No. 2011/0253224, filed Apr. 15, 2011, entitled“Feedback Control in Microfluidic Systems,” [C1256.70006US01]; U.S.Patent Publication No. 2011/0256551, filed Apr. 15, 2011, entitled“Systems and Devices for Analysis of Samples,” [C1256.70010US01], eachof which is incorporated herein by reference in its entirety for allpurposes.

EXAMPLES Example 1

This example shows the influence of channel geometry and surface tensionof a fluid on volume reduction of a fluid plug during flow of the fluidplug in a channel.

Fluid plugs containing a fluid and varying concentrations of wettingagent were flowed through several channels that differed only inhydraulic diameter. The volume reduction of a fluid plug for a givenchannel length increased as the hydraulic diameter of the channelincreased. The volume reduction for a given channel length alsoincreased as the surface tension decreased (i.e., as the amount ofwetting agent in the fluid plug increased). The effect of hydraulicdiameter was less pronounced for fluids as the surface tensiondecreased.

Three fluid plugs, varying only in the concentration of wetting agent,were flowed through channels with a hydraulic diameter of 0.4 mm to 1.0mm. The distance required for complete volume reduction of each fluidplug (i.e., length of channel required to disperse the plug in mm permicroliters) was recorded for each hydraulic diameter. Polyvinyl alcoholwas used as the wetting agent to reduce the surface tension of thefluid. Each fluid plug contained 0.025% polyvinyl alcohol, 0.08%polyvinyl alcohol, or 0.4% polyvinyl alcohol in deionized water. FIG. 8shows the distance required for each concentration of polyvinyl alcoholfor each hydraulic diameter tested.

This example demonstrates that the volume reduction of a fluid plug(and, therefore, the amount of mixing between fluids) can be varied bytailoring the channel geometry and/or the surface tension of the fluidcontained in the fluid plug.

Example 2

This example shows the influence of surface energy of the channel andsurface tension of the fluid on volume reduction.

Two identical channels with a height of 3.5 mm and a width of 0.5 mmwere fabricated. One channel was treated with atmospheric coronadischarge to increase the surface energy of the channel. A coronadischarge was applied for about 1 second at a distance of 1 cm away fromthe surface of the channel. The corona discharge treatment produced asurface energy of greater than 72 dynes/cm as indicated by deionizedwater spreading into a film rather than beading up. Polyvinyl alcoholwas used as the wetting agent to reduce the surface tension of thefluid. The fluid plugs contained either 0.025% polyvinyl alcohol or0.08% polyvinyl alcohol in deionized water. FIG. 9 shows the length ofchannel required for complete volume reduction of the fluid plug foreach concentration of polyvinyl alcohol for the untreated and thecorona-treated channel.

Two channels that differed only in their surface energy were formed.Fluid plugs containing a fluid and varying concentrations of wettingagent were flowed through the two channels. The volume reduction of afluid plug, for a given channel length, increased as surface energyincreased. The volume reduction, for a given channel length, alsoincreased as the surface tension decreased (i.e., as the amount ofwetting agent in the fluid plug increased). The effect of surface energywas less pronounced with decreased surface tension. In addition, theeffect of surface tension was less pronounced with increased surfaceenergy.

The corona treated channel had about a 50% decrease in mean length ofchannel required to disperse the plug compared to the untreated channelfor a fluid plug with 0.025% polyvinyl alcohol. Decreasing the surfacetension of the fluid caused about a 50% decrease in mean length ofchannel required to disperse the plug in the untreated channel.

This example demonstrates that the volume reduction of a fluid plug(and, therefore, the amount of mixing between fluids) can be varied bytailoring the surface energy of the channel containing the fluid plug,and/or the surface tension of the fluid contained in the fluid plug.

Example 3

This example shows serial mixing of multiple fluid plugs in a channel.

A microfluidic channel was loaded with fluid plugs containing air or asolution of deionized water containing 5 mg/mL of a blue dye (methyleneblue) or 10 mg/mL of a red dye (allura red). The aqueous fluid plugswere immiscible with the air fluid plugs. The fluid plugs alternatedbetween aqueous fluid plugs and air fluid plugs. The aqueous fluid plugsalternated in dye color, such that the first aqueous fluid plugcontained a red dye, the second contained a blue dye, third contained ared dye, etc. The channel contained nine fluid plugs of each dye color.Each aqueous fluid plug had a volume of 2 μL. Mixing was initiated byconnecting a vacuum of approximately 30 kPa to the outlet of the system.From the outlet, the mixed solutions flowed through a microfluidicchannel in which the optical density of the solutions was measured withred and green light as described below. The ratio of red dye to blue dyein the fluid plugs after flowing in the channel could be calculated fromthese measurements using the regression model described above.

The optical densities of the aqueous fluid plugs were measured afterfollowing in the channel. An LED emitting either red (˜630 nm) or green(˜505 nm) light was positioned above the channel, while an opticaldetector was positioned below the channel, and the optical transmissionthrough the channel was monitored and recorded using a data capturesystem. The optical density was calculated using the opticaltransmission of deionized water without any dye as a reference value.Various solutions containing known concentrations of red dye, blue dye,or a mixture of the two were flowed through the system, and the opticaldensity was measured with red and green light. A multivariate regressionmodel was fit to these results to permit estimation of the dyeconcentration in mixed solutions based on the optical density measuredwith red and green light. This model was used in the experimentsdescribed herein.

Mixing of multiple fluid plugs was shown using aqueous fluid plugsdiffering only in the presence of either a red dye or a blue dye. Theaqueous fluid plugs alternated in dye color and were separated by fluidplugs containing air. The fluid plugs were flowed in a microfluidicchannel. The ratios of red and blue dye in each aqueous fluid plug weremeasured after flowing in the microfluidic channel for about 350 mm (ie, the first plug was 350 mm away from their initial positions). Thefirst three aqueous fluid plugs were completely dissipated along thechannel wall, as indicated by the absence of any data points in fluidplug #s 1-3 in FIG. 10, and were absorbed by the subsequent aqueousfluid plugs. After the sixth aqueous fluid plug, the percent of red dyeand blue dye in each aqueous fluid plug was within 50%±5% which was theinitial overall percentage of red and blue dye in the channel. FIG. 10shows a plot of the percent of the red and blue dye in each aqueousfluid plug calculated from their measured optical densities afterflowing in the microfluidic system. The overall enrichment of redsolution in the earlier segments is attributed to the fact that thefirst and third aqueous fluid plugs were red.

This example demonstrates that serial mixing can be performed withmultiple fluid plugs in a channel. This example also demonstrates thatthe ratio of components in the fluid plugs after mixing converges towardthe initial overall ratio of components in the total volume loaded intothe channel.

Example 4

This example shows the influence of surface roughness and channel lengthon mixing of multiple fluid plugs.

Mixing of multiple fluid plugs was performed with an identical set-up asExample 3, except the microfluidic channel had an additional length of630 mm and the additional channel length had been treated withmicro-abrasive blasting to change the surface texture. Roughness wasmeasured by stylus profilometry with a stylus tip radius ofapproximately 2 μm. The channel had an average roughness between about0.1 μm and 0.5 μm. The first four aqueous fluid plugs were completelydissipated along the channel wall. After the fifth aqueous fluid plug,the ratio of red dye to blue dye in the aqueous fluid plugs was about50:50, which was the initial overall ratio of red dye to blue dye.

FIG. 11 shows a plot of the percent of the red and blue dye in eachaqueous fluid plug calculated from their measured optical densitiesafter flowing in the microfluidic system. The first four aqueous fluidplugs were distributed along the channel, and were absorbed by thesubsequent aqueous fluid plugs. After the fifth aqueous fluid plug, thepercent of red dye and blue dye was within 50%±5% in each fluid plug.The overall enrichment of red solution in the earlier segments isattributed to the fact that the first and third aqueous fluid plugs werered.

This example demonstrates that surface roughness can be used to increasefluid dissipation and enhance mixing in a channel.

Example 5

This example shows the influence of volume of the fluid plugs on mixingof multiple fluid plugs.

Mixing of multiple fluid plugs was performed with an identical set-up asExample 3, except the first aqueous fluid plug had a volume of 1microliter. The first and second aqueous fluid plugs were completelydissipated along the channel wall. After the second aqueous fluid plug,the ratio of red dye to blue dye in the aqueous fluid plugs was about50:50.

FIG. 12 shows a plot of the percent of the red and blue dye in eachaqueous fluid plug calculated from their measured optical densitiesafter flowing in the microfluidic channel. The first and second aqueousfluid plugs were entirely dissipated along the channel, and wereabsorbed by the subsequent aqueous fluid plugs. After the second aqueousfluid plug, the percent of red dye and blue dye in each fluid plug waswithin 50%±5%.

This example demonstrates that the ratio of components in each fluidplug after mixing is dependent upon the volume of the fluid plugscarrying the fluids to be mixed.

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. Accordingly, the foregoing description and drawings areby way of example only.

What is claimed is:
 1. A method, comprising: flowing in a channel aseries of fluid plugs comprising a first fluid plug comprising a firstfluid, a second fluid plug comprising a second fluid, and a third fluidplug comprising a third fluid, wherein the first fluid plug has a firstvolume, wherein the second fluid plug is positioned between the firstand third fluid plugs, and wherein the second fluid is immiscible witheach of the first and third fluids, the method comprising: while thefirst fluid plug is flowing, depositing at least a portion of the firstfluid on a wall of the channel for combining with the third fluid plug;reducing the first volume of the first fluid plug by at least 50%; andcombining at least a portion of the first fluid into the third fluidplug so as to mix at least portions of the first and third fluids, andafter the combining step, flowing in the channel the first fluid plug,the second fluid plug, and the third fluid plug which comprises thethird fluid and at least a portion of the first fluid.
 2. A method,comprising: flowing in series in a channel a first fluid plug comprisinga first fluid, a second fluid plug comprising a second fluid, and athird fluid plug comprising a third fluid, wherein the second fluid isimmiscible with each of the first and third fluids, wherein the secondfluid plug is positioned between the first and third fluid plugs, andwherein the first fluid comprises a first component for a chemicaland/or biological reaction and the third fluid comprises a secondcomponent for a chemical and/or biological reaction, and wherein thefirst component is different from the second component; and while thefirst fluid plug is flowing, depositing at least a portion of the firstfluid on a wall of the channel for combining with the third fluid plug;and combining at least a portion of the first fluid deposited on thewall of the channel into the third fluid plug so as to mix at leastportions of the first and third fluids, and after the combining step,flowing in the channel the first fluid plug, the second fluid plug, andthe third fluid plug which comprises the third fluid and at least aportion of the first fluid.
 3. A method, comprising: flowing in seriesin a channel a first fluid plug comprising a first fluid, a second fluidplug comprising a second fluid, and a third fluid plug comprising athird fluid, wherein the first fluid comprises a first component for achemical and/or biological reaction and the third fluid comprises asecond component for a chemical and/or biological reaction, wherein thesecond fluid is immiscible with the first and third fluids, and whereinthe second fluid plug is positioned between the first and third fluidplugs; and while the first fluid plug is flowing, depositing at least aportion of the first fluid on a wall of the channel for combining withthe third fluid plug, and combining at least a portion of the firstfluid into the third fluid plug so as to mix at least portions of thefirst and third fluids, and after the combining step, flowing in thechannel the first fluid plug, the second fluid plug, and the third fluidplug which comprises the third fluid and at least a portion of the firstfluid; and performing one or more chemical and/or biological reactionsinvolving each of the first and second components.
 4. A method as inclaim 1, comprising reducing the first volume of the first fluid plug byat least 75%.
 5. A method as in claim 1, comprising reducing the firstvolume of the first fluid plug by 100% such that the first fluid isentirely dispersed within the channel.
 6. A method as in claim 1,wherein, the first fluid and the third fluid are miscible with oneanother.
 7. A method as in claim 3, comprising performing a chemicaland/or biological reaction between the first and second components.
 8. Amethod as in claim 1, flowing in series a fourth fluid plug comprising afourth fluid and a fifth fluid plug comprising a fifth fluid along withthe first, second and third fluid plugs, wherein the fourth fluid isimmiscible with the first, third and fifth fluids.
 9. A method as inclaim 1, comprising reducing the volume of the third fluid plug.
 10. Amethod as in claim 1, comprising reducing the volume of the third fluidplug by at least 50%.
 11. A method as in claim 8, comprising combiningat least a portion of the third fluid into the fifth fluid plug so as tomix at least portions of the third and fifth fluids.
 12. A method as inclaim 1, wherein the first and/or third fluid comprises a component fora chemical and/or biological reaction.
 13. A method as in claim 1,wherein the first and/or third fluid comprises a wetting agent.
 14. Amethod as in claim 1, wherein the first and/or third fluid comprises ametal salt.
 15. A method as in claim 1, wherein the second fluidcomprises a gas.
 16. A method as in claim 1, wherein the second fluid ishydrophobic.
 17. A method as in claim 1, wherein the second fluid isdirectly adjacent the first and third fluids.
 18. A method as in claim1, wherein the channel has a cross-section comprising a radius ofcurvature smaller than the half-width of the channel.
 19. A method as inclaim 1, wherein the channel has a cross-section comprising a radius ofcurvature smaller than the half-height of the channel.
 20. A method asin claim 1, wherein the channel has an average diameter of less than orequal to about 1 mm.
 21. A method as in claim 1, wherein the channel,prior to the flowing step, has been subjected to plasma treatment.
 22. Amethod as in claim 1, wherein prior to the flowing step, the firstand/or third fluids are stored and sealed in the channel or in one ormore branching channels in fluid communication with the channel.
 23. Amethod as in claim 1, wherein the first fluid is deposited as a film onthe wall of the channel.
 24. A method as in claim 1, wherein the firstfluid is deposited as fluid droplets on the wall of the channel.
 25. Amethod as in claim 1, wherein the combining step occurs while the firstfluid plug, the second fluid plug, and the third fluid plug are flowingin series.
 26. A method as in claim 1, wherein the channel is configuredto remove at least a portion of the first fluid from the first fluidplug during the step of flowing.